A central model for experimental carcinogenesis is the two-stage hypothesis, in which cancer induction by prolonged exposure to a arcinogen is assumed to be replaceable by limited, subcarcinogenic exposure followed by promotion by a (usually) nonmutagenic promoter. The latter regimen has been realized in several systems, is assumed by many to be a model for carcinogenesis generally, and is considered to be a possible major contributor to the risk of cancer in man. Neither of the latter assumptions or considerations has been adequately tested experimentally, and we hypothesize that they are invalid. Tests of this hypothesis will be carried out in mouse skin and rat liver, the two systems best characterized to date. In mouse skin, the specific hypothesis to be pursued is that papilloma and carcinoma induction proceed by separate progression pathways. Testing will be used on tumor induction kinetics using the promoters 12-O-tetradecanoylphorbol-13-acetate and 7-bromomethylbenz[a]anthracene, the promotion inhibitor fluocinolone acetonide, and the induction of autonomous and promoter-dependent papillomas with different regimens. Biochemical markers (Gamma-glutamyltranspeptidase, fucosyltransferase, ornithine decarboxylase, methyleation of cytosine) will be used to characterize different cell populations and tumors. In rat liver, attempts will be made to develop a system in which tumors can be induced by a series of events clearly separated in time, in order to take advantage of liver's relatively quiescent state. Under these conditions it may be possible to show that events associated with promotion in mouse skin take place during the events required for initiation in rat liver. Development of a potential new system will be begun, initiation and promotion of mammary tumors in the male rat, based on recent findings with 2-acetamidophenanthrene and DDT. Finally, the rat liver system will be exploited in an attempt to define unambiguously the role of 8-(n-2-fluor-enylacetamido)guanine adducts on DNA in the carcinogenic activity of 2-acetamidofluorene, by determination of the effect of sulfotransferase inhibitor on the initiating activity of AAF. The proposed project is thus expected to shed light on the relationship of chemical binding, mutagenesis, and tumor initiation to each other, and on the role of promotion in the induction of human cancer.